The pentose phosphate pathway (PPP) is an important pathway for protecting cells from oxidative stress. The PPP will typically be stimulated in response to oxygen radical formation from such things as exposure to xenobiotics and radiation.
The oxidative branch of the pentose phosphate pathway produces ribose-5-P, CO.sub.2 and NADPH from glucose-6-phosphate (Glc-6-P) and NADP.sup.+ and water. GIc-6-P is converted from glucose by the enzyme hexokinase. (See FIG. 2 for synthesis scheme.) Katz, J. et al., J. Biol Chem. 235:2165-2177 (1960) and Wood, H. G. et al., Biochem. Z. 338:809-847 (1963). NADPH can be used for lipid synthesis and antioxidant defense, and ribose-5-P can be used for nucleotide formation. The ribose-5-P can also be converted by the nonoxidative branch of the PPP to fructose-6-P (Fru-6-P) and glyceraldehyde-3-P, which can then be converted to lactate by glycolytic enzymes. Fru-6-P can also recycle through the PPP. Alternatively, lactate is produced via glycolysis without following the PPP, specifically, Glc-6-P (converted from glucose) can convert to Fru-6-P and glyceraldehyde-3-P without first converting to ribose-5-P. Since both pathways utilize Glc-6-P of the PPP, and ribose-5-P of the PPP converts to Fru-6 -P of the glycolytic pathway, the pathways are considered coupled.
Activity of the PPP is commonly quantitated by measuring .sup.14 CO.sub.2 production from the C1 of [1-.sup.14 C]glucose. A parallel incubation with [6-.sup.14 C]glucose is required to correct for .sup.14 CO.sub.2 production from the citric acid cycle. Katz, J. et al., J. Biol. Chem. 235:2165-2177 (1960); Wood, H. G. et al., Biochem. Z. 338:809-847 (1963); Katz, J. et al., J. Biol. Chem. 241:727-740. (1966) and Hothersall, J. S. et al., Arch. Biochem. Biophys. 198:478-492 (1979). This method has various limitations such as extensive Krebs cycle activity producing large amounts of .sup.14 CO.sub.2 from both [1-.sup.14 C]glucose and [6-.sup.14 C]glucose, thus making it difficult to quantitate low levels of PPP activity. Jolley, R. L. et al., J. Biol. Chem. 233:1289-1294 (1958), In addition, only a single measurement can be made on each sample because the sample must be destroyed by acidification to release CO.sub.2. Two parallel incubations with [1 -.sup.14 C]glucose and [6-.sup.14 C]glucose must also be performed for each measurement with intersample variability increasing the uncertainty of the data. Furthermore, calculations of PPP activity require measuring the amount of glucose consumed. Wood, H. G. et al., Biochem. Z. 338:809-847 (1963); Katz, J. et al., J. Biol. Chem. 241:727-740 (1966) and Hothersall, J. S. et al., Arch. Biochem. Biophys. 198:478-492 (1979).
Although the first two limitations can be overcome by measuring the isotopic composition of triose phosphate derivatives such as lactate rather than CO.sub.2 (Katz, J. et al., J. Biol. Chem. 235:2165-2177 (1960); Wood, H. G. et al., Biochem. Z. 338:809-847 (1963) and Katz, J. et al., J. Biol. Chem. 241:727-740 (1966)), efforts to quantitate PPP activity in a single incubation by measuring (3-.sup.13 C)lactate and (3-.sup.12 C)lactate produced from (1-.sup.13 C)glucose have been complicated by an unknown amount of (3-.sup.12 C)lactate produced from endogenous (unlabeled) substrates. Willis, J. A. et al., Biochem. Biophys. Res. Commun. 138:1068-1073 (1986); Cheng, H-M. et al., J. Ocular Pharmacol. 2:319-324 (1986); Ross, B. D. et al. NMR Biomed. 1:20-26 (1988); Mitchell, S. L. et al., Biochem. Biophys. Res. Commun. 158:474-479 (1989) and Miceli, M. V. et al., Invest. Opthalmol. Vis. Sci. 277-283 (1990). Thus, a parallel incubation with (6-.sup.13 C)glucose is required. Kingsley-Hickman, P. B. et al., Anal. Biochem. 185:235-237 (990).
It would thus be desirable to provide an isotope and method for measuring or assaying the activities of the glycolysis and pentose phosphate pathway (PPP). It would also be desirable to provide an isotope and method for measuring the relative activities of glycolysis and the PPP which only requires a single incubation. It would further be desirable to provide an isotope and method for measuring glycolysis and PPP activity wherein repeated measurements can be made on a single sample. It would also be desirable to provide an isotope and method for measuring glycolysis and PPP activity which is not interfered by the presence of extensive CO.sub.2. It would further be desirable to provide an isotope and method for measuring relative activities of glycolysis and the PPP which does not require the measurement of consumed glucose.